Fig 2: PM2.5-promoted tumor growth and metastasis are associated with IL-17a in xenograft mouse models. (A) Representative images of tumor samples isolated from each group of mice as indicated (n = 6). Scale bar, 1 cm. (B) Tumor volume was measured (n = 6). (C) Tumor weight was recorded (n = 6). (D) The body weight of mice was recorded (n = 6). (E) IHC staining was used to determine KI-67 and SOX2 expression levels in the tumor sections (n = 4). Scale bar, 100 μm. (F) H&E staining of pulmonary tissues (n = 4; Scale bar, 100 μm) and (G) pictures of lung samples isolated from the indicated groups of mice to calculate the metastatic nodules on the surface of lungs (n = 6). (H) The number of lung metastatic nodules was quantified (n = 6). (I) RT-qPCR analysis was used to calculate the expression of metastasis-associated genes as displayed (n = 4). All data are expressed as mean ± SEM. *p<0.05 and **p<0.01.

Fig 3: PM2.5 elevates the proliferation in NSCLC cells. (A–E) Th17 cells were treated with 100 μg/cm2 of PM2.5 for 24 h, and then all cells and supernatants were collected for the analysis. (A) TNF-α and IL-6 levels in the supernatants were measured using ELISA. (B) IL-17a contents in the collected supernatants were calculated using ELISA. (C) RT-qPCR and (D) western blot analysis were used to measure IL-17a expression levels in the harvested cells. (E) IF staining of IL-17a in the harvested cells. Scale bar, 20 μm. (F) Th17 cells were treated with 100 μg/cm2 of PM2.5 for 24 h, and then the conditional medium was collected, and mixed with fresh RPMI1640 absolute medium at 1:3. The composed culture medium was exposed to A549 and H1350 cells for 12, 24, 48 or 72 h. Then, the NSCLC cells were collected for cell proliferation analysis using CCK-8 analysis. (G, H) Th17 cells were incubated with 100 μg/cm2 of PM2.5 for 24 h, and then the conditional medium was collected, and mixed with fresh RPMI1640 absolute medium at 1:3. Then, the composed culture medium was subjected to A549 and H1350 cells for another 24 h. Subsequently, all cells were harvested to assess the cell proliferation using colony formation and EdU assays. Scale bar, 100 μm. Quantification of colony formation assay and EdU was exhibited. All data are expressed as mean ± SEM. *p<0.05 and **p<0.01 compared to the Ctrl group.

Fig 4: IL-17a treatment promotes the proliferation and EMT in NSCLC cells. (A–G) A549 and H1350 cells were treated with or without IL-17a (100 ng/ml) for 24 h, and then were collected for the following studies. (A) CCK-8 analysis was used to determine the cell proliferative activity. (B) Colony formation and EdU assays were used to determine the cell proliferative condition. Scale bar, 100 μm. (C) Quantification of the number of cells by colony formation and EdU analysis. (D) Transwell analysis was used to calculate the number of cells in migration and invasion. Scale bar, 100 μm. (E) Quantification of the counts of the migrated and invaded cells. (F) RT-qPCR analysis was used to measure EMT-associated genes in A549 and H1350 cells. (G) IF staining of N-cadherin in A549 and H1350 cells. Scale bar, 50 μm. All data are expressed as mean ± SEM. *p<0.05 and **p<0.01 compared to the Ctrl group.

Fig 5: IL-17a knockout alleviates pulmonary injury and cancer stem cell properties in mice following PM2.5 exposure for 3 months. (A) H&E staining (up panel) and Masson trichrome staining (down panel) of lung sections from IL-17a+/+ and IL-17a-/- mice challenged with or without PM2.5 for 3 months (n = 6). Scale bar, 100 μm. (B) Protein levels in BALF were measured (n = 8). (C) The total number of cells in BALF was assessed (n = 8). (D) The number of neutrophils in BALF was measured (n = 8). (E) Serum TNF-α and IL-6 levels in mice were measured by ELISA (n = 8). (F) TNF-α and IL-6 mRNA levels in the pulmonary samples were determined using RT-qPCR analysis (n = 4). (G) Fibrosis-associated genes as shown were tested using RT-qPCR analysis (n = 4). (H) Genes associated with lung cancer progression were calculated using RT-qPCR analysis (n = 4). (I) IHC staining of c-Myc and SOX2 in pulmonary sections from the indicated groups of mice (n = 6). Scale bar, 100 μm. All data are expressed as mean ± SEM. *p<0.05 and **p<0.01 compared to the FA/IL-17a+/+ group; +p<0.05 compared to the PM2.5/IL-17a+/+ group.